EKSTRAKSI, purifikasi dan PRINSIP KLONING DNA Agustina Setiawati, M.Sc., Apt

EKSTRAKSI DNA Why we do it

What do we need DNA for? Forensik/DNA profiling Kloning Diagnosis penyakit Sekuensing DNA Genetically modified organism (GMO) Pengujian lingkungan

Sistematika ekstraksi dan purifikasi DNA Penumbuhan sel Panen sel dan lisis Pemekatan DNA Purifikasi DNA

1. Ekstraksi sel Butuh reagen LYSIS detergen Buffer enzim protease panas “cell extract”

DETERGEN Melarutkan lipid pada membran sel CTAB (hexadecyltrimethylammonium bromide) – sel tumbuhan Laurylsarcosine—bakteri gram negative

DETERGEN Soap molecules and grease molecules are made of two parts: Heads, which like water Tails, which hate water.

DETERGEN Sel mempunyai membran lipid double layer dan protein.

DETERGEN When detergent comes close to the cell, it captures the lipids and proteins.

Enzim protease—Proteinase K Menghilangkan nuclear protein, enzim Memecah ikatan peptida Bisa ditambahkan atau tidak (optional) Panas Paling sering 40-60ºC Buffer Tris HCl pH 8 untuk menjaga stabilitas DNA

2. Penghilangan protein & RNA a. Ekstrak sel dicampur dengan fenol, perlahan! b. Tambahkan Rnase pada lapisan aqueous

3. Pemekatan konsentrasi DNA Sentrifugasi 70% final conc. “spooling” Ethanol precipitation

ANALISIS KUANTITATIF DNA Absorbansi/Optical density (OD): c x b x (extinction coefficient, E). Asam nukleat mengabsorbsi sinar UV pada 260 nm       1 A260 O.D. unit for dsDNA = 50 µg/ml       1 A260 O.D. unit for ssDNA = 33 or 50 µg/ml       1 A260 O.D. unit for RNA = 40 µg/ml

Spectrophotometric analysis of DNA

Double-stranded and single-stranded DNA differ in their optical absorption at 260 nm dC dG dU dsDNA ssDNA nucleotides The conjugated p-electron systems of the purine & pyrimidine bases absorb strongly in the UV. (That’s why UV light is mutagenic and carcinogenic.) The absorbance of double-stranded DNA (dsDNA) at 260 nm is less than that of either single-stranded DNA (ssDNA) or the free bases. This is called “hypochromism.”

Kemurnian DNA The purity of the DNA is reflected in the OD260:OD 280 ratio and must be between 1.6 and 2.00. Decreased 260:280 ratio means that contaminating protein is still present.

ANALISIS KUALTITAIF Metode: Elektroforesis Gel Agarose buffer   Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer. Cathode (negative) Anode (positive)  wells

Agarose is a linear polymer extracted from seaweed. D-galactose 3,6-anhydro L-galactose Sweetened agarose gels have been eaten in the Far East since the 17th century. Agarose was first used in biology when Robert Koch* used it as a culture medium for Tuberculosis bacteria in 1882 *Lina Hesse, technician and illustrator for a colleague of Koch was the first to suggest agar for use in culturing bacteria Agarose is a linear polymer extracted from seaweed. PRAKTIKUM BIOLOGI MOLEKULER, 2007

+ - DNA H  O2  Power • DNA is negatively charged. • When placed in an electrical field, DNA will migrate toward the positive pole (anode). H  O2 • An agarose gel is used to slow the movement of DNA and separate by size. + - Power Scanning Electron Micrograph of Agarose Gel (1×1 µm)  • Polymerized agarose is porous, allowing for the movement of DNA

+ - How fast will the DNA migrate? DNA small large Power strength of the electrical field, buffer, density of agarose gel… Size of the DNA! *Small DNA move faster than large DNA …gel electrophoresis separates DNA according to size DNA + - Power small large Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.

VISUALISASI DNA

Teknologi dna rekombinan Agustina Setiawati, M.Sc., Apt

Kloning DNA Cloning – the act of making many identical copies of a particular piece of DNA (often a gene) As you know, the first stop often involves joining a piece of DNA of interest to a cloning vector using DNA ligase

Prinsip DNA rekombinan Penyisipan DNA target ke dalam vektor Memasukkan DNA rekombinan dalam bakteri

TAHAP KLONING Pemilihan vektor Pemotongan vektor dan DNA target dengan enzim retriksi Penyambungan DNA pada vektor Transformasi Seleksi hasil transformasi

VEKTOR KLONING

Pemilihan Vektor Syarat vektor: Kecil Mempunyai gen spesifik untuk penanda Punya retriksi untuk beberapa enzim retriksi Origin of Replication (ORI)

Plasmid DNA untai ganda sirkuler, ekstrakromosom Linier : Streptomyces rochei Ukuran : 2,2 kb – 700 kb Jumlah duplikat: 1-2; 4-8; 20-30, 700-1000.

Penamaan plasmid p : plasmid BR : pembuat Bolivar dan Rodriguez 322 dibuat lebih awal drpd pBR325, pBR328

VEKTOR PLASMID pBr322

VEKTOR PLASMID puc18/19

Keuntungan pUC Jumlah duplikat 500 – 700 plasmid/sel Mudah mendeteksi % plasmid rekombinan Adanya polycloning sites pUC18 = pUC19, polycloning sitesnya berlawanan Membawa promoter lacUV dan ribosome binding site

Pemotongan DNA & vektor

PEMOTONGAN HpaI

PEMOTONGAN EcoRI

Ligasi

LIGASI

Transformasi

TRANSFORMASI

Electroporation Prinsip: pembukaan membran pembentukan pori sel tanaman dengan muatan listrik DNA in the surrounding solution can enter the cell through these pores and become incorporated into the cell’s nuclear genome through illegitimate recombination

Biolistic transformation – “Gene gun” DNA is precipitated on the surface of heavy metal (gold; tungsten) particles Loaded particles are driven into plant cells by high velocity gas propulsion (originally gunpowder; now helium) Distance between discharge nozzle and tissue can be optimized, as can particle velocity Target tissue must be regenerable

Seleksi Transformasi

pUC18/19

Deteksi adanya klon yang diinginkan pada pUC

Seleksi pUC18/19 Sel yang membawa plasmid (non rekombinan dan rekombinan) resisten terhadap ampisilin. Sel yang punya plasmid non rekombinan menghasilkan warna biru pada medium yang mengandung laktosa

Plasmid Polylinkers and Marker Genes for Blue-White screening A vector usually contains a sequence (polylinker) which can recognize several restriction enzymes so that the vector can be used for cloning a variety of DNA samples. Colonies with recombinant plasmids are white, and colonies with nonrecombinant plasmids are blue. Example: pUC19 Resistant to ampicillin, has (ampr gene) Contains portion of the lac operon which codes for beta- galactosidase. X-gal is a substrate of beta-galactosidase and turns blue in the presence of functional beta-galactosidase is added to the medium. Insertion of foreign DNA into the polylinker disrupts the lac operon, beta-galactosidase becomes non-functional and the colonies fail to turn blue, but appear white. DNA rekombinan pada plasmid

pBR322

KLONING DENGAN VEKTOR pBR322

KEMUNGKINAN BERHASIL PERSAMAA CLARCK DAN CARBON N = ln(1 – P)/ln(1 – F) P : probalitas 0,99 F : ukuran sisipan/ukuran kromosom N : jumlah koloni dg plasmid rekombinan

Koloni yang dibutuhkan Ukuran kromosom 4.000 kb Ukuran rata-rata sisipan 7,7 kb N=ln(1-99)/ln(1-7,7/4000) = 2390 koloni Manusia 4x106 kb

Kloning gena isulin manusia Ukuran kromosom 4x109 pb Ukuran gena isulin 1.700 pb Ukuran sisipan 10.000 pb N = ln(1-0,99)/ln(1-104/4x109) Jika sisipan 40.000 pb N = ?

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